The LAT52 gene of tomato is expressed in a pollen-specific manner. It is shown that LAT52 encodes a heat-stable, glycosylated protein that traverses the secretory pathway when expressed in a baculovirus expression system. The LAT52 protein shows some similarity with Kunitz trypsin inhibitors and with pollen proteins from maize, rice and olive, but the biological function of these pollen proteins is unknown. To test whether the LAT52 protein plays an important role during pollen development, tomato plants were transformed with an antisense LAT52 gene driven by the LAT52 promoter. Because the LAT52 gene is expressed gametophytically, only 50% of the pollen of the primary transformants would be expected to express the antisense construct. Selfprogeny of 19 of the primary transformants showed the predicted 3:1 segregation for a single locus insertion of the linked kanamycin-resistance gene. However, the self-progeny of the other 32 primary transformants showed a 1:1 segregation pattern and could not transmit the linked kanamycin-resistance gene through the male. A subset of these 1:1 segregation class plants was examined in detail. The pollen showed lower levels of LAT52 mRNA and LAT52 protein when compared with wild-type. In vitro, approximately 50% of the pollen grains appear to hydrate abnormally; this anomaly is not present when the same pollen grains are incubated in a medium with higher water potential. In vivo pollination experiments showed that the growth of around 50% of the pollen tubes is arrested in the style. The 3:1 segregation class plants showed no significant differences from untransformed control plants. Taken together, the results show a direct correlation between the reduced expression of LAT52 protein and abnormal pollen function, and suggest that the LAT52 protein plays a role in pollen hydration and/or pollen germination.